1. Field of the Invention
The present invention relates to a method for non-invasive local quantification of angio-genesis or elimination of existing blood vessels in living tissue, to the use of near infrared spectroscopy and/or laser doppler flowmetry for local measurements of hemoglobin concentration and blood perfusion at the same location on the subject, and to an apparatus for non-invasive quantification of angiogenesis or elimination of blood vessels in living tissue.
2. Background Art
In normal tissue many important processes such as wound healing, tissue growth and the development of the fetus during pregnancy involves the formation of new blood vessels (capillaries). This process of vascularisation also known as angiogenesis is of paramount importance since supply of nutrients including oxygen as well as removal of waste products constitute an absolute prerequisite for survival and growth.
In cancerous tissue, tumors cannot grow or spread (metastasize) without the development of new blood vessels. Solid tumors cannot grow beyond the size of a pin-head (1 to 2 cubic millimeters) without inducing the formation of new blood vessels to supply the nutritional needs of the tumor.
Endothelial cells, that form the walls of blood vessels, are the source of new blood vessels. The creation of new blood vessels occurs by a series of sequential steps whereby the endothelial cells forming the wall of an existing small blood vessel (capillary) become activated and start to invade the matrix creating new networks of blood vessels which makes tissue growth possible. New capillary growth is tightly controlled by a finely tuned balance between factors that activate endothelial cell growth and those that inhibit it.
In recent years the process of angiogenesis has been realized as a potential target for anti-tumor growth since any therapy which will prevent the angiogenesis will inherently restrict tumor growth.
Since hemoglobin is a strong absorber of light, it has been known for a long time that hemoglobin concentration can be quantified by spectroscopic techniques. Also since the absorption of hemoglobin changes during oxygenation/deoxygenation it is well known that the oxygenation state of hemoglobin in tissue may be quantified. The absorption spectrum of hemoglobin and deoxyhemoglobin is characteristically different also in the near infrared region of the spectrum (700-1100 nm), where the absorption in water (and hence in tissue) is relatively small. This has led to the development of NIRS (near infrared spectroscopy) of tissues, with instrumentation by which it is possible to evaluate the oxygenation of a given tissue region (e.g. Brazy et al. 1985, Pediatrics 75:217-225; Chance et al. 1988, Anal. Biochem. 174:698-707).
As concluded in a review by R. K. Jain et al (Nature Medicine, 1997,3: 1203-1208), a prerequisite to answering important questions about the biology of angiogenesis and to effectively seek out new therapeutic agents, with beneficial effects on angiogenesis, is the availability of a quantitative, rapid and routine angiogenesis assay. Furthermore the authors have stated, that such an assay has not been currently available.
Existing in vivo assays can be divided into three categories: Exteriorized tissue preparations, chronic transparent chambers and other in situ preparations including artificial matrix implants, and excised tissues. Overall the assays were only quantitative at the expense of the tissue integrity, and thus forfeiting the potential for repeated measurements (monitoring). Existing assays, on the other hand, are either not quantitative or they are based on quite complicated micro surgical procedures, which are prohibitively expensive and too time-consuming for screening purposes.
Selective blocking of each of the six principal steps in the angiogenic cascade can lead to inhibition or halting of tumor growth. Since the mid-1990ties many pharmaceutical companies involved in cancer therapeutics have focused intensely on the development of anti-angiogenic compounds, and approximately 30 compounds have already been through various phases of clinical testing. In parallel, a series of related compounds, which by different mechanisms destroy existing blood vessels in tumors are under development. This category of experimental cancer drugs is called vascular targeted agents. They destroy existing vasculature and make tumors shrink, whereas anti-angiogenic agents obstruct further growth and spread of small tumors by preventing the formation of essential new vascular networks.
In order to develop and test new cancer therapeutic agents it is desirable to provide a fast, reproducible method which makes it possible to monitor changes in angiogenesis or destruction of blood vessels versus time in response to application or administration of anti-angiogenic therapeutic agents or vascular targeting agents to a subject. This requires a non-invasive method which will not affect the viability of the localized tissue cells. Since angiogenesis involves the formation of new capillaries, measurement of changes versus time of total hemoglobin in a specific location will also provide a quantitative or at least semi-quantitative measure of local angiogenesis. Likewise, the formation of new blood vessels will result in a local increase in blood perfusion versus time.
Accordingly it is an object of the present invention to provide a simple method for measuring the relative local changes in hemoglobin concentration and/or blood perfusion. The method shall be non-invasive and non-harmful to the tissue and the subject. In this way measurements can be performed on the same subject repeatedly and for long periods of time. In order to save time consuming measurements involving separate measurements for each parameter and at the same time ensure that both parameters to be measured are done so at the exact same location on the tissue of the subject it is also an object of the invention to provide an apparatus facilitating simultaneous measurement of NIRS (near infrared spectroscopy) and LDF (laser doppler flowmetry) at the same location. According to a first aspect of the invention, these and other objects are accomplished by providing a method for non-invasive local quantification of angiogenesis or quantification of elimination of existing blood vessels in living tissue comprising near infrared spectroscopy (NRS) for measuring total hemoglobin concentration and/or Laser Doppler Flowmetry (LDF) for measuring the average blood perfusion of the same location.
The invention also provides a method for local non-invasive measurement of hemoglobin concentration and blood perfusion at the same location on the subject after application or administration of a substance chosen from the group comprising anti-angiogenic agents, vascular targeting agents, anti-inflammatory agents, skin transplants, or any agent that will affect blood circulation. The method consists of a near infrared spectroscopy (NIRS) measurement, and a laser doppler flowmetry (LDF) measurement performed at the same location and substantially at the same time. The performed measurements of NIRS and LDF can be continuously repeated in order to monitor the time dependent response after application or administration of the above agents.
According to a second aspect the invention also relates to an apparatus for non invasive quantification of angiogenesis or quantification of destruction of existing blood vessels in living tissue comprising a xenon flash unit, an optical filter, and a Y-shaped optical fiber bundle, one branch of the fibre bundle being coupled to the flash unit and the other branch of the fiber bundle being coupled to the detection unit, while the merged part of the fibre bundle may be coupled to the probe of the LDF equipment in such a way that the NIRS and the LDF measurements originate from nearly the same tissue location when the merged NIRS/LDF probe is placed on the tissue surface.
According to a third aspect of the invention there is also provided a use of near infrared spectroscopy and laser doppler flowmetry for local measurements of hemoglobin concentration and blood perfusion at the same location on the subject after application or administration of a substance chosen from the group comprising, anti-angiogenic agents, vascular targeted agents, skin agents, skin transplants, or any agent that will affect blood circulation.